Human B-cell translocation genes-2 and 3 antibodies

ABSTRACT

The present invention relates to novel antiproliferative genes. More specifically, isolated nucleic acid molecules are provided encoding the human B-cell translocation genes 2 and 3 (BTG-2 and BTG-3). BTG-2 and BTG-3 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same.

[0001] The present application is a continuation-in-part (CIP) of U.S.patent application Ser. No. 08/463,382, filed Jun. 5, 1995, whichdisclosure is herein incorporated by reference. The present applicationis also a CIP of U.S. patent application Ser. No. 08/460,104, filed Jun.2, 1995, which disclosure is herein incorporated by reference. U.S.patent application Ser. Nos. 08/463,382 and 08/460,104 claim prioritybenefit under 35 U.S.C. §120 to PCT/US95/03323, filed Mar. 17, 199-5,which disclosure is herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to novel antiproliferative genes.More specifically, isolated nucleic acid molecules are provided encodingthe human B-cell translocation genes 2 and 3 (BTG-2 and BTG-3). BTG-2and BTG-3 polypeptides are also provided, as are vectors, host cells andrecombinant methods for producing the same.

[0004] 2. Related Art

[0005] In normal tissues, homeostasis is maintained through negative andpositive growth controls which effect the proliferation anddifferentiation related to cellular genetic programs. An alteration ofthis subtle balance can result in developmental abnormalities or inneoplasia. Proto-oncogenes, genes that promote cell division, were thefirst growth-inducing elements to be identified and more than sixty ofthem have been described so far (Bishop, J. M., Cell 64:235:248 (1991)).The genes that negatively regulate cell proliferation are crucial tocounteract the growth-inducing elements and are likely to have the sameimportance as proto-oncogenes in controlling cell division (Marshall, C.J., Cell 64:313-326 (1991)), especially since the loss of their functionhas been reported to be associated with irregular cellulardifferentiation and proliferation or with alteration of embryonicdevelopment (Weinberg, R. A., Science 254:1138-1146 (1991)).

[0006] The polynucleotides and polypeptides of the present invention arethought to be members of a family of anti-proliferative genes. BTG-1 isa member of this group and has been cloned and expressed. (Rovault, J.P. et al., The EMBO Journal 11(4):1663-1670 (1992)). BTG-1 was shown tonegatively regulate N1H3T3 cell proliferation when over- orinappropriately expressed. BTG stands for B-cell translocation gene, andthe BTG-1 gene has been shown to be involved in a chromosomaltranslocation [t(8;12)(q24;22)] in B-cell chronic lymphocytic leukemia.

[0007] The BTG-1 open reading frame is 60% homologous to PC3, animmediate early gene induced by nerve growth factor in rat PC12 cells.Sequence and Northern blot analyses indicate that BTG-1 and PC3 are notcognate genes but are thought to be members of this new family ofanti-proliferation genes. The BTG-1 gene is preferentially expressed inquiescent cells during the early sub-phases of G₁ in a serum-dependentmanner and it is then down-regulated to reach a minimum level as thecells enter the S phase. This suggests a functional link between BTG-1and the cell cycle process. BTG-1 is expressed in tissues (lymphoid,liver, placenta) containing non-dividing cells likely to re-enter thecell cycle upon different stimuli, whereas the expression of BTG-1 isbarely detectable in fully differentiated tissues such as brain andmuscle.

[0008] The BTG-1 gene was shown to be highly conserved in evolution anda similar 1.8 Kb transcript can be detected in murine and chicken tissueby using a human BTG-1 DNA probe (Rimokh, R. et al., Genes Chrom. Cancer3:24-36 (1991)).

[0009] The BTG-2 and BTG-3 genes and gene products have been putativelyidentified as members of this family as a result of amino acid sequencehomology to BTG-1.

SUMMARY OF THE INVENTION

[0010] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the BTG-2 or BTG-3 polypeptidehaving the amino acid sequences shown in FIG. 1 (SEQ ID NO:2) and FIG. 2(SEQ ID NO:4), respectively, or having the amino acid sequences encodedby the cDNA clones deposited in bacterial hosts as ATCC Deposit Number97025 on Jan. 17, 1995 (BTG-2) and ATCC Deposit Number 97010 on Jan. 5,1995 (BTG-3). For BTG-2, the nucleotide sequence determined bysequencing the deposited cDNA clone contains an open reading frameencoding a polypeptide of about 345 amino acid residues, with apredicted leader sequence of about 25 amino acid residues such that themature protein comprises about 320 amino acids. The amino acid sequenceof the predicted mature BTG-2 protein is shown in FIG. 1, amino acidresidues from about 26 to about 345 (SEQ ID NO:2).

[0011] For BTG-3, the nucleotide sequence determined by sequencing thedeposited cDNA clone contains an open reading frame encoding apolypeptide of about 344 amino acid residues, with a predicted leadersequence of about 18 amino acid residues such that the mature proteincomprises about 326 amino acids. The amino acid sequence of thepredicted mature BTG-3 protein is shown in FIG. 2, amino acid residuesfrom about 19 to about 344 (SEQ ID NO:4).

[0012] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the BTG-2 or BTG-3 polypeptide having the complete amino acidsequence in FIG. 1 (SEQ ID NO:2) and FIG. 2 (SEQ ID NO :4),respectively; (b) a nucleotide sequence encoding the mature BTG-2 ormature BTG-3 polypeptide having the amino acid sequence at positionsfrom about 26 to about 345 in FIG. 1 (SEQ ID NO:2) and from about 19 toabout 344 in FIG. 2 (SEQ ID NO:4), respectively; (c) a nucleotidesequence encoding the BTG-2 or BTG-3 polypeptide having the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97025 and 97010, respectively; (d) a nucleotide sequence encodingthe mature BTG-2 or mature BTG-3 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No.97025and 97010, respectively; and (e) a nucleotide sequence complementary toany of the nucleotide sequences in (a), (b), (c) or (d) above.

[0013] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably at least 95%, 96%,97%, 98% or 99% identical, to any of the nucleotide sequences in (a),(b), (c), (d) or (e), above, or a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide in (a), (b), (c),(d) or (e), above. This polynucleotide which hybridizes does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues. An additional nucleic acid embodiment of the invention relatesto an isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of a BTG-2or BTG-3 polypeptide having an amino acid sequence in (a), (b), (c) or(d), above.

[0014] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of BTG-2 or BTG-3 polypeptides or peptides by recombinanttechniques.

[0015] The invention further provides an isolated BTG-2 or BTG-3polypeptide having an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of the BTG-2 or BTG-3polypeptide having the complete 345 and 344 amino acid sequences shownin FIG. 1 (SEQ ID NO:2) and FIG. 2 (SEQ ID NO:4), respectively; (b) theamino acid sequence of the predicted mature BTG-2 or BTG-3 polypeptide(without the leader) having the amino acid sequence at positions fromabout 26 to about 345 in FIG. 1 (SEQ ID NO:2) and from about 19 to about344 in FIG. 2 (SEQ ID NO:4), respectively; (c) the amino acid sequenceof the BTG-2 or BTG-3 polypeptide having the complete amino acidsequence, including the leader, encoded by the cDNA clone contained inATCC Deposit No. 97025 and 97010, respectively; and (d) the amino acidsequence of the mature BTG-2 or BTG-3 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97025and 97010, respectively. The polypeptides of the present invention alsoinclude polypeptides having an amino acid sequence with at least 90%similarity, and more preferably at least 95% similarity to thosedescribed in (a), (b), (c) or (d) above, as well as polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those above.

[0016] An additional embodiment of this aspect of the invention relatesto a peptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a BTG-2 or BTG-3 polypeptide having an aminoacid sequence described in (a), (b), (c) or (d), above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a BTG-2 or BTG-3 polypeptide of the invention includeportions of such polypeptides with at least six or seven, preferably atleast nine, and more preferably at least about 30 amino acids to about50 amino acids, although epitope-bearing polypeptides of any length upto and including the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention.

[0017] In another embodiment, the invention provides an isolatedantibody that binds specifically to a BTG-2 or BTG-3 polypeptide havingan amino acid sequence described in (a), (b), (c) or (d) above. Suchantibodies are useful diagnostically or therapeutically as describebelow.

[0018] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, to treat disease states characterized by aberrant cellularproliferation, and to modulate cellular growth.

[0019] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, in thetreatment of diseases related to chromosomal translocation, for example,lymphocytic leukemia.

[0020] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting diseases related tothe under-expression of the polypeptides of the present invention andmutations in the nucleic acid sequences encoding such polypeptides.

[0021] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 is an illustration of the cDNA and corresponding deducedamino acid sequence of BTG-2. One-letter abbreviations for amino acidsare used. Sequencing was performed, for both BTG-2 and BTG-3, using a373 Automated DNA sequencer (Applied Biosystems, Inc.). Sequencingaccuracy is predicted to be greater than 97% accurate.

[0023]FIG. 2 shows the cDNA and corresponding deduced amino acidsequence of the putative BTG-3.

[0024]FIG. 3 is an amino acid sequence alignment between BTG-1, BTG-2and BTG-3 proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a BTG-2 or BTG-3 polypeptide havingthe amino acid sequence shown in FIG. 1 (SEQ ID NO:2) and FIG. 2 (SEQ IDNO:4), respectively, which were determined by sequencing cloned cDNAs.The BTG-2 and BTG-3 proteins of the present invention share sequencehomology with each other and with BTG-1 (FIG. 3) (SEQ ID NO:5 (BTG-Isequence) and SEQ ID NO:6 (consensus sequence)). The nucleotidesequences shown in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3) wereobtained by sequencing the above described cDNA clones, which weredeposited at the American Type Culture Collection, 12301 Park LawnDrive, Rockville, Md. 20852, and given the accession numbers indicatedabove. The deposited clones are contained in the pBluescript SK(−)plasmid (Stratagene, LaJolla, Calif.).

Nucleic Acid Molecules

[0026] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0027] Unless otherwise indicated, each “nucleotide sequence” set forthherein is presented as a sequence of deoxyribonucleotides (abbreviatedA, G, C and T). However, by “nucleotide sequence” of a nucleic acidmolecule or polynucleotide is intended, for a DNA molecule orpolynucleotide, a sequence of deoxyribonucleotides, and for an RNAmolecule or polynucleotide, the corresponding sequence ofribonucleotides (A, G, C and U), where each thymidinedeoxyribonucleotide (T) in the specified deoxyribonucleotide sequence isreplaced by the ribonucleotide uridine (U). For instance, reference toan RNA molecule having the sequence of SEQ ID NO:1 or SEQ ID NO:3 setforth using deoxyribonucleotide abbreviations is intended to indicate anRNA molecule having a sequence in which each deoxyribonucleotide A, G orC of SEQ ID NO:1 or SEQ ID NO:3 has been replaced by the correspondingribonucleotide A, G or C, and each deoxyribonucleotide T has beenreplaced by a ribonucleotide U.

[0028] Using the information provided herein, such as the nucleotidesequence in FIG. 1 or FIG. 3, a nucleic acid molecule of the presentinvention encoding a BTG-2 or BTG-3 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecules described in FIG. 1 (SEQ ID NO:1) and FIG. 2(SEQ ID NO:3) were discovered in cDNA libraries derived from a humanendometrial tumor and a human synovial carcinoma, respectively. ForBTG-2, the gene can also be identified in cDNA libraries from thefollowing tissues: liver, lymphoid and placenta. For BTG-3, the gene canalso be identified in cDNA libraries from the following tissues:synovial sarcoma, cerebellum, embryonic, and placenta.

[0029] The determined nucleotide sequence of the BTG-2 cDNA of FIG. 1(SEQ ID NO:1) contains an open reading frame encoding a protein of about345 amino acid residues and a predicted leader sequence of about 25amino acid residues. The amino acid sequence of the predicted matureBTG-2 protein is shown in FIG. 1 (SEQ ID NO:2), amino acid residue fromabout 26 to about 345. The BTG-2 protein shown in FIG. 1 (SEQ ID NO:2)is about 49% identical and about 72% similar to BTG-1 over a 91 aminoacid stretch (FIG. 3).

[0030] The determined nucleotide sequence of the BTG-3 cDNA of FIG. 2(SEQ ID NO:3) contains an open reading frame encoding a protein of about344 amino acid residues and a predicted leader sequence of about 18amino acid residues. The amino acid sequence of the predicted matureBTG-3 protein is shown in FIG. 2 (SEQ ID NO:4), amino acid residue fromabout 19 to about 344. The BTG-3 protein shown in FIG. 2 (SEQ ID NO:4)is about 41% identical and about 74% similar to BTG-1 over an 85 aminoacid stretch (FIG. 3). Over the same stretch of 85 amino acids, BTG-2and BTG-3 are 83% identical and 87% similar to each other. In addition,BTG-2 is approximately 143 amino acids longer at the carboxy terminus ascompared to BTG-1, while BTG-3 is approximately 162 amino acids longerat the carboxy terminus. Both BTG-2 and BTG-3 contain unique regionsrich in the amino acids proline and glutamine.

[0031] As indicated, the present invention also provides mature forms ofthe BTG-2 and BTG-3 proteins. According to the signal hypothesis,proteins secreted by mammalian cells have a signal or secretory leadersequence which is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Most mammalian cells and even insect cells cleave secretedproteins with the same specificity. However, in some cases, cleavage ofa secreted protein is not entirely uniform, which results in two or moremature species on the protein. Further, it has long been known that thecleavage specificity of a secreted protein is ultimately determined bythe primary structure of the complete protein, that is, it is inherentin the amino acid sequence of the polypeptide. Therefore, the presentinvention provides nucleotide sequences encoding the mature BTG-2 andBTG-3 polypeptides having the amino acid sequences encoded by the cDNAclones contained in the hosts identified as ATCC Deposit No. 97025 and97010, respectively.

[0032] By the mature BTG-2 and BTG-3 polypeptides having the amino acidsequences encoded by the deposited cDNA clones is meant the mature formsof these proteins produced by expression in a mammalian cell (e.g., COScells, as described below) of the complete open reading frame encoded bythe human DNA,sequence of the clones contained in the vectors in thedeposited hosts. As indicated below, the mature BTG-2 and BTG-3polypeptides having the amino acid sequences encoded by the depostedcDNA clones may or may not differ from the predicted “mature” BTG-2 andBTG-3 polypeptides shown in FIG. 1 and FIG. 2, respectively. Thisdepends on the accuracy of the predicted cleavage site based on computeranalysis.

[0033] Methods for predicting whether a protein has a secretory leaderas well as the cleavage point for that leader sequence are availablebecause it is known that much of the cleavage specificity for asecretory protein resides in certain amino acid residues within thesignal sequence and the N-terminus of the mature protein, particularlyresidues immediately surrounding the cleavage site. For instance, themethod of McGeoch (Virus Res. 3:271-286 (1985)) uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje (NucleicAcids Res. 14:46834690 (1986)) uses the information from the residuessurrounding the cleavage site, typically residues -13 to +2 where +1indicates the amino acid terminus of the mature protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. von Heinje, supra.However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

[0034] Thus, in view of above, as one of ordinary skill wouldappreciate, the actual leader sequence of the BTG-2 protein of thepresent invention is predicted to be about 25 amino acids in length, butmay be anywhere in the range of about 15 to about 35 amino acids.Similarly, the actual leader sequence of the BTG-3 protein of thepresent invention is predicted to be about 18 amino acids in length, butmay be anywhere in the range of about 8 to about 28 amino acids.Further, due to possible sequencing errors as discussed above, thefull-length BTG-2 protein is predicted to be about 345 amino acids inlength, but may be in the range of about 335 to about 355 amino acids.Similarly, the full-length BTG-3 protein is predicted to be about 344amino acids in length, but may be in the range of about 335 to about 355amino acids.

[0035] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0036] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0037] Isolated nucleic acid molecules of the present invention includeDNA molecules shown in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3);DNA molecules comprising the coding sequence for the mature BTG-2 andBTG-3 proteins shown in FIG. 1 (SEQ ID NO:2) and FIG. 2 (SEQ ID NO:3),respectively; and DNA molecules which comprise a sequence substantiallydifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode the BTG-2 or BTG-3 protein. Of course,the genetic code is well known in the art. Thus, it would be routine forone skilled in the art to generate such degenerate variants.

[0038] In another aspect, the invention provides isolated nucleic acidmolecules encoding the BTG-2 or BTG-3 polypeptide having an amino acidsequence encoded by the above-described deposited cDNA clones.Preferably, these nucleic acid molecules will encode the maturepolypeptides encoded by the deposited cDNA clones. The invention furtherprovides isolated nucleic acid molecules having a sequence complementaryto one of the above sequences. Such isolated molecules, particularly DNAmolecules, are useful as probes for gene mapping, by in situhybridization with chromosomes, and for detecting expression of theBTG-2 or BTG-3 gene in human tissue, for instance, by Northern blotanalysis.

[0039] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of one ofthe deposited cDNAs or the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1) or FIG. 2 (SEQ ID NO:2) is intended fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt inlength which are useful as diagnostic probes and primers as discussedherein. Of course, larger fragments 50-1500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequences of the deposited cDNAs oras shown in FIG. 1 (SEQ ID NO:1) or FIG. 2 (SEQ ID NO:2). By a fragmentat least 20 nt in length, for example, is intended fragments whichinclude 20 or more contiguous bases from the nucleotide sequences of thedeposited cDNA or the nucleotide sequence as shown in FIG. 1 (SEQ IDNO:1) or FIG. 2 (SEQ ID NO:2). Since the BTG-2 and BTG-3 genes have beendeposited and the nucleotide sequences shown in FIG. 1 (SEQ ID NO:1) andFIG. 2 (SEQ ID NO:3) are provided, generating such DNA fragments wouldbe routine to the skilled artisan. For example, restriction endonucleasecleavage or shearing by sonication could easily be used to generatefragments of various sizes. Alternatively, such fragments could begenerated synthetically.

[0040] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the BTG-2 orBTG-3 protein. Methods for generating such epitope-bearing portions ofthe BTG-2 or BTG-3 protein are described in detail below.

[0041] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone contained in ATCC Deposit No. 97025 or 97010. By“stringent hybridization conditions” is intended overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0042] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0043] Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50-750 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited cDNAs or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1) or FIG. 2 (SEQ ID NO:3). By aportion of a polynucleotide of “at least 20 nt in length,” for example,is intended 20 or more contiguous nucleotides from the nucleotidesequence of the reference polynucleotide. As indicated, such portionsare useful diagnostically either as a probe according to conventionalDNA hybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), the entire disclosureof which is hereby incorporated herein by reference. Of course, apolynucleotide which hybridizes only to a poly A sequence (such as the3′ terminal poly(A) tract of the BTG-2 or BTG-3 cDNA shown in FIG. 1(SEQ ID NO:1)) and FIG. 2 (SEQ ID NO:2), respectively, or to acomplementary stretch of T (or U) resides, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

[0044] As indicated, nucleic acid molecules of the present inventionwhich encode a BTG-2 or BTG-3 polypeptide may include, but are notlimited to those encoding the amino acid sequence of the maturepolypeptide, by itself; the coding sequence for the mature polypeptideand additional sequences, such as those encoding the leader or secretorysequence, such as a pre-, or pro- or prepro-protein sequence; the codingsequence of the mature polypeptide, with or without the aforementionedadditional coding sequences, together with additional, non-codingsequences, including for example, but not limited to introns andnon-coding 5′ and 3′ sequences, such as the transcribed, non-translatedsequences that play a role in transcription, mRNA processing, includingsplicing and polyadenylation signals, for example—ribosome binding andstability of mRNA; an additional coding sequence which codes foradditional amino acids, such as those which provide additionalfunctionalities. Thus, the sequence encoding the polypeptide may befused to a marker sequence, such as a sequence encoding a peptide whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (Qiagen, Inc.), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37: 767 (1984). As discussed below, other such fusion proteinsinclude the BTG-2 or BTG-2 protein fused to Fc at the N- or C-terminus.

[0045] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the BTG-2 or BTG-3 protein. Variants may occurnaturally, such as a natural allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985). Non-naturally occurring variants may beproduced using art-known mutagenesis techniques.

[0046] Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the BTG-2 or BTG-3 protein or portionsthereof. Also especially preferred in this regard are conservativesubstitutions.

[0047] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 90% identical, and more preferably at least 95%, 96%, 97%, 98%or 99% identical to: (a) a nucleotide sequence encoding the BTG-2 orBTG-3 polypeptide having the complete amino acid sequence in FIG. 1 (SEQID NO:2) and FIG. 2 (SEQ ID NO:4), respectively; (b) a nucleotidesequence encoding the mature BTG-2 or mature BTG-3 polypeptide havingthe amino acid sequence at positions from about 26 to about 345 in FIG.1 (SEQ ID NO:2) and from about 19 to about 344 in FIG. 2 (SEQ ID NO:4),respectively; (c) a nucleotide sequence encoding the BTG-2 or BTG-3polypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97025 and 97010, respectively; (d) anucleotide sequence encoding the mature BTG-2 or mature BTG-3polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No.97025 and 97010, respectively; and (e) anucleotide sequence complementary to any of the nucleotide sequences in(a), (b), (c) or (d) above.

[0048] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aBTG-2 or BTG-3 polypeptide is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence encoding theBTG-2 or BTG-3 polypeptide. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0049] As a practical matter, whether any particular nucleic acidmolecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIG. 1 or FIG. 3 or to thenucleotide sequence of one of the deposited cDNA clones can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711. Bestfit uses the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), tofind the best segment of homology between two sequences. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

[0050] The present application is directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIG. 1 (SEQ ID NO:1) or FIG. 3 (SEQ ID NO:3) or to thenucleic acid sequence of one of the deposited cDNAs, irrespective ofwhether they encode a polypeptide having BTG-2 or BTG-3 activity. Thisis because even where a particular nucleic acid molecule does not encodea polypeptide having BTG-2 or BTG-3 activity, one of skill in the artwould still know how to use the nucleic acid molecule, for instance, asa hybridization probe or a polymerase chain reaction (PCR) primer. Usesof the nucleic acid molecules of the present invention that do notencode a polypeptide having BTG-2 or BTG-3 activity include, inter alia,(1) isolating the BTG-2 or BTG-3 gene or allelic variants thereof in acDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of the BTG-2or BTG-3 gene, as described in Verma et al., Human Chromosomes: A Manualof Basic Techniques, Pergamon Press, New York (1988); and Northern Blotanalysis for detecting BTG-2 or BTG-3 mRNA expression in specifictissues.

[0051] Preferred, however, are nucleic acid molecules having sequencesat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIG. 1 (SEQ ID NO:1) or FIG. 2 (SEQ ID NO:3) or to thenucleic acid sequence of one of the deposited cDNAs which do, in fact,encode a polypeptide having BTG-2 or BTG-3 protein activity. By “apolypeptide having BTG-2 or BTG-3 activity” is intended polypeptidesexhibiting activity similar, but not necessarily identical, to anactivity of the BTG-2 or BTG-3 protein of the invention (either thefull-length protein or, preferably, the mature protein), as measured ina particular biological assay. For example, the BTG-2 and BTG-3 proteinsare antiproliferative agents. In fact, exogenously expressed BTG-2protein has been shown to suppress growth of NIH3T3 cells (Matsuda etal., Oncogene 12:705-713 (1996)). Thus, BTG-2 or BTG-3 protein activitycan be assayed by measuring the ability of a candidate polypeptide tosuppress growth of certain cell types in vitro, such as NIH3T3 cells.

[0052] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA or the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) willencode a polypeptide “having BTG-2 or BTG-3 protein activity.” In fact,since degenerate variants of these nucleotide sequences all encode thesame polypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving BTG-2 or BTG-3 protein activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid).

[0053] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main approaches for studying the toleranceof an amino acid sequence to change. The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein.

Vectors and Host Cells

[0054] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof BTG-2 and BTG-3 polypeptides or fragments thereof by recombinanttechniques.

[0055] Recombinant constructs may be introduced into host cells usingwell known techniques such infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0056] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0057] Preferred are vectors comprising cis-acting control regions tothe polynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

[0058] In certain preferred embodiments in this regard, the vectorsprovide for specific expression, which may be inducible and/or celltype-specific. Particularly preferred among such vectors are thoseinducible by environmental factors that are easy to manipulate, such astemperature and nutrient additives.

[0059] Expression vectors useful in the present invention includechromosomal-, episomal- and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, bacteriophage, yeast episomes, yeastchromosomal elements, viruses such as baculoviruses, papova viruses,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

[0060] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

[0061] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS and Bowes melanoma cells; and plant cells.Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

[0062] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0063] Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

[0064] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0065] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0066] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

[0067] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5- has been fused with Fc portions for the purpose of high-throughputscreening assays to identify antagonists of hIL-5. See, D. Bennett etal., Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K.Johanson et al., The Journal of Biological Chemistry, Vol. 270, No.16:9459-9471 (1995).

[0068] The BTG-2 and BTG-3 proteins can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

BTG-2 and BTG-3 Polypeptides and Fragments

[0069] The invention further provides an isolated BTG-2 or BTG-3polypeptide having the amino acid sequence encoded by the depositedcDNAs, or the amino acid sequence in FIG. 1 (SEQ ID NO:2) or FIG. 2 (SEQID NO:4), or a peptide or polypeptide comprising a portion of the abovepolypeptides. The terms “peptide” and “oligopeptide” are consideredsynonymous (as is commonly recognized) and each term can be usedinterchangeably as the context requires to indicate a chain of at leastto amino acids coupled by peptidyl linkages. The word “polypeptide” isused herein for chains containing more than ten amino acid residues. Alloligopeptide and polypeptide formulas or sequences herein are writtenfrom left to right and in the direction from amino terminus to carboxyterminus.

[0070] It will be recognized in the art that some amino acid sequencesof the BTG-2 or BTG-3 polypeptide can be varied without significanteffect of the structure or function of the protein. If such differencesin sequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity. In general, itis possible to replace residues which form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

[0071] Thus, the invention further includes variations of the BTG-2 orBTG-3 polypeptide which show substantial BTG-2 or BTG-3 polypeptideactivity or which include regions of BTG-2 or BTG-3 protein such as theprotein portions discussed below. Such mutants include deletions,insertions, inversions, repeats, and type substitutions (for example,substituting one hydrophilic residue for another, but not stronglyhydrophilic for strongly hydrophobic as a rule). Small changes or such“neutral” amino acid substitutions will generally have little effect onactivity.

[0072] Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchangeof the acidic residues Asp and Glu, substitution between the amideresidues Asn and Gln, exchange of the basic residues Lys and Arg andreplacements among the aromatic residues Phe, Tyr. Examples ofconservative amino acid substitutions known to those skilled in the artare set forth below: Aromatic: phenylalanine tryptophan tyrosineHydrophobic: leucine isoleucine valine Polar: glutamine asparagineBasic: arginine lysine histidine Acidic: aspartic acid glutamic acidSmall: alanine serine threonine methionine glycine

[0073] As indicated in detail above, further guidance concerning whichamino acid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

[0074] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of the BTG-2 or BTG-3 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

[0075] The polypeptides of the present invention include the BTG-2 orBTG-3 polypeptide encoded by the deposited cDNA clones (ATCC Nos. 97025and 97010, respectively) including the leader, the mature polypeptideencoded by the deposited the cDNA clones minus the leader (i.e., themature protein), the BTG-2 polypeptide of FIG. 1 (SEQ ID NO:2) includingthe leader, the BTG-3 polypeptide of FIG. 2 (SEQ ID NO:4) including theleader, the BTG-2 polypeptide of FIG. 1 (SEQ ID NO:2) minus the leader,the BTG-3 polypeptide of FIG. 2 (SEQ ID NO:4) minus the leader, as wellas polypeptides which have at least 90% similarity, more preferably atleast 95% similarity, and still more preferably at least 96%, 97%, 98%or 99% similarity to those described above. Further polypeptides of thepresent invention include polypeptides at least 80% identical, morepreferably at least 90% or 95% identical, still more preferably at least96%, 97%, 98% or 99% identical to the BTG-2 or BTG-3 polypeptide encodedby the deposited cDNA clones (ATCC Nos. 97025 and 97010, respectively),to the BTG-2 polypeptide of FIG. 1 (SEQ ID NO:2), to the BTG-3polypeptide of FIG. 2 (SEQ ID NO:4) and also include portions of suchBTG-2 or BTG-3 polypeptides with at least 30 amino acids and morepreferably at least 50 amino acids.

[0076] By “% similarity” for two polypeptides is intended a similarityscore produced by comparing the amino acid sequences of the twopolypeptides using the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) and the defaultsettings for determining similarity. Bestfit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics2:482489, 1981) to find the best segment of similarity between twosequences.

[0077] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a BTG-2or BTG-3 polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the BTG-2 or BTG-3polypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0078] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIG. 1 (SEQ ID NO:2), FIG. 2 (SEQ ID NO:4)or to the amino acid sequence encoded by deposited cDNA clones can bedetermined conventionally using known computer programs such the Bestfitprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science Drive,Madison, Wis. 53711. When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

[0079] The BTG-2 or BTG-3 polypeptide of the present invention could beused as a molecular weight marker on SDS-PAGE gels or on molecular sievegel filtration columns using methods well known to those of skill in theart.

[0080] As described in detail below, the polypeptides of the presentinvention can also be used to raise polyclonal and monoclonalantibodies, which are useful in assays for detecting BTG-2 or BTG-3protein expression as described below or as agonists and antagonistscapable of enhancing or inhibiting BTG-2 or BTG-3 protein function.Further, such polypeptides can be used in the yeast two-hybrid system to“capture” BTG-2 or BTG-3 protein binding proteins which are alsocandidate agonist and antagonist according to the present invention. Theyeast two hybrid system is described in Fields and Song, Nature340:245-246 (1989).

[0081] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide of the invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. These immunogenicepitopes are believed to be confined to a few loci on the molecule. Onthe other hand, a region of a protein molecule to which an antibody canbind is defined as an “antigenic epitope.” The number of immunogenicepitopes of a protein generally is less than the number of antigenicepitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983).

[0082] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies thatreact with predetermined sites on proteins. Science 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Peptides that are extremelyhydrophobic and those of six or fewer residues generally are ineffectiveat inducing antibodies that bind to the mimicked protein; longer,peptides, especially those containing proline residues, usually areeffective. Sutcliffe et al., supra, at 661. For instance, 18 of 20peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

[0083] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.Thus, a high proportion of hybridomas obtained by fusion of spleen cellsfrom donors immunized with an antigen epitope-bearing peptide generallysecrete antibody reactive with the native protein. Sutcliffe et al.,supra, at 663. The antibodies raised by antigenic epitope-bearingpeptides or polypeptides are useful to detect the mimicked protein, andantibodies to different peptides may be used for tracking the fate ofvarious regions of a protein precursor which undergoespost-translational processing. The peptides and anti-peptide antibodiesmay be used in a variety of qualitative or quantitative assays for themimicked protein, for instance in competition assays since it has beenshown that even short peptides (e.g., about 9 amino acids) can bind anddisplace the larger peptides in immunoprecipitation assays. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

[0084] Antigenic epitope-bearing peptides and polypeptides of theinvention designed according to the above guidelines preferably containa sequence of at least seven, more preferably at least nine and mostpreferably between about 15 to about 30 amino acids contained within theamino acid sequence of a polypeptide of the invention. However, peptidesor polypeptides comprising a larger portion of an amino acid sequence ofa polypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

[0085] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means for making peptides orpolypeptides including recombinant means using nucleic acid molecules ofthe invention. For instance, a short epitope-bearing amino acid sequencemay be fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) General methodfor the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods. A completely manual procedure allows 500-1000 or more synthesesto be conducted simultaneously. Houghten et al., supra, at 5134.

[0086] Epitope-bearing peptides and polypeptides of the invention areused to induce antibodies according to methods well known in the art.See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow,M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. etal., J. Gen. Virol. 66:2347-2354 (1985). Generally, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling of the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine may be coupled to carrier using a linkersuch as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 g peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

[0087] Immunogenic epitope-bearing peptides of the invention, i.e.,those parts of a protein that elicit an antibody response when the wholeprotein is the immunogen, are identified according to methods known inthe art. For instance, Geysen et al., supra, discloses a procedure forrapid concurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

[0088] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C -alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

[0089] The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

[0090] As one of skill in the art will appreciate, BTG-2 or BTG-3polypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature331:84- 86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric BTG-2 or BTG-3 proteinor protein fragment alone (Fountoulakis et al., J Biochem 270:3958-3964(1995)).

Diagnostic and Therapuetic Uses

[0091] The BTG-2 and BTG-3 polypeptides have an anti-proliferativeability and may be employed to treat diseases or pathological conditionsassociated with aberrant cellular proliferation. The polypeptides may beemployed as a tumor/growth suppression regulator. They may also beemployed to inhibit cancer cell proliferation.

[0092] BTG-2 and BTG-3 may also be employed to prevent uncontrolledwound healing which would otherwise cause scarring. Restenosis, which isre-occlusion of arterial walls after balloon angioplasty, may also betreated with BTG-2 and BTG-3 since arteries re-occlude through cellproliferation. Similarly angiogenesis of tumors may be inhibited.

[0093] The BTG-2 and BTG-3 genes and gene products may also be employedfor modulation of cellular growth. Due to their anti-proliferativeeffect they could be selectively administered or possibly inhibited whenit is desirable to have certain cells proliferate. An example would be adisorder related to the underproduction of certain cells, whereproliferation and differentiation of these cells would help to treat thedisorder.

[0094] The polynucleotides and polypeptides encoded by suchpolynucleotides may also be utilized for in vitro purposes related toscientific research, synthesis of DNA and manufacture of DNA vectors andfor designing therapeutics and diagnostics for the treatment of humandisease.

[0095] The nucleic acid sequences of the present invention may beemployed as part of a diagnostic assay for detecting susceptibility todiseases associated with aberrant cellular proliferation. Since, thepolypeptides of the present invention are anti-proliferative genes, adisruption in the transcription of the genes and corresponding lack ofproduction of the gene product will likely be involved in aberrantcellular proliferation associated with a malignant phenotype.

[0096] Individuals carrying mutations in the genes may be detected atthe DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, including but not limited toblood, urine, saliva, tissue biopsy and autopsy material. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature 324:163-166 (1986)) prior toanalysis. RNA or cDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid encoding thepolypeptides of the present invention can be used to identify andanalyze mutations. For example, deletions and insertions can be detectedby a change in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA of the present invention or alternatively,radiolabeled antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0097] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

[0098] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and SI protection or thechemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci.(USA) 85:4397-4401 (1985)).

[0099] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0100] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0101] The present invention also relates to a diagnostic assay fordetecting altered levels of the proteins of the present invention invarious tissues since an over-expression of the proteins compared tonormal control tissue samples may detect the presence of a disease orsusceptibility to a disease, for example, abnormal cellularproliferation and differentiation. Assays used to detect levels of theseproteins in a sample derived from a host are well-known to those ofskill in the art and include radioimmunoassays, competitive-bindingassays, Western Blot analysis, ELISA assays and “sandwich” assay. AnELISA assay (Coligan et al., Current Protocols in Immunology 1(2),Chapter 6, (1991)) initially comprises preparing an antibody specific tothe antigens to the polypeptides of the present invention, preferably amonoclonal antibody. In addition a reporter antibody is prepared againstthe monoclonal antibody. To the reporter antibody is attached adetectable reagent such as radioactivity, fluorescence or, in thisexample, a horseradish peroxidase enzyme. A sample is removed from ahost and incubated on a solid support, e.g. a polystyrene dish, thatbinds the proteins in the sample. Any free protein binding sites on thedish are then covered by incubating with a non-specific protein, forexample, bovine serum albumen. Next, the monoclonal antibody specific tothe polypeptides of the present invention is incubated in the dishduring which time the monoclonal antibodies attach to any proteinsattached to the polystyrene dish. All unbound monoclonal antibody iswashed out with buffer. The reporter antibody linked to horseradishperoxidase is now placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to the proteins ofthe present invention. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount ofprotein present in a given volume of patient sample when comparedagainst a standard curve.

[0102] A competition assay may be employed wherein antibodies specificto the polypeptides of the present invention are attached to a solidsupport. Labeled polypeptides and a sample derived from the host arethen passed over the solid support and the amount of label detected, forexample by liquid scintillation chromatography, can be correlated to aquantity of the polypeptides of the present invention in the sample.

[0103] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay the polypeptides of the present invention are passed over a solidsupport and bind to antibodies attached to a solid support. A secondantibody is then bound to the polypeptides. A third antibody which islabeled and specific to the second antibody is then passed over thesolid support and binds to the second antibody and an amount can then bequantified.

[0104] This invention provides a method for identification of thereceptors for the polypeptides of the present invention. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting(Coligan, et al., Current Protocols in Immun. 1(2), Chapter 5, (1991)).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, and a cDNA librarycreated from this RNA is divided into pools and used to transfect COScells or other cells that are not responsive to the polypeptides.Transfected cells which are grown on glass slides are exposed to thelabeled polypeptides. The polypeptides can be labeled by a variety ofmeans including iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to auto-radiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aniterative sub-pooling and re-screening process, eventually yielding asingle clones that encodes the putative receptor.

[0105] As an alternative approach for receptor identification, thelabeled polypeptides can be photo-affinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicro-sequencing. The amino acid sequence obtained from micro-sequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Agonists and Antagonists

[0106] The present invention further provides a method of identifyingagonist and antagonist compounds to the genes and gene products of thepresent invention. An example of such an assay comprises contacting amammalian cell or membrane preparation expressing the receptors of thepolypeptides with labeled polypeptides, eg. by radioactivity, in thepresence of a compound to be screened. The ability of the compound toblock and enhance the interaction of the polypeptides of the presentinvention with its receptor is then measured, for example, by liquidscintillation chromatography.

[0107] This invention provides a method of screening drugs to identifythose which enhance or inhibit interaction of the polypeptides withtheir receptors. As an example, a mammalian cell or membrane preparationexpressing the receptor would be incubated with labeled polypeptides inthe presence of the drug. The ability of the drug to enhance or blockthis interaction could then be measured.

[0108] Alternatively, the response of a known second messenger systemfollowing interaction of the polypeptides and their receptors would bemeasured and compared in the presence or absence of the drug. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

[0109] Potential antagonists to BTG-2 or BTG-3 polypeptides include anantibody, or in some cases, an oligopeptide, which binds to thepolypeptide. Alternatively, a potential antagonist may be a closelyrelated protein which binds to the receptor sites, however, they areinactive forms of the polypeptide and thereby prevent their action sincereceptor sites are occupied.

[0110] Another potential antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix -see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al,Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)),thereby preventing transcription and the production of BTG-2 and/orBTG-3. The antisense RNA oligonucleotide hybridizes to the mRNA in vivoand blocks translation of the mRNA molecule into the polypeptides(Antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of the polypeptides of the present invention.

[0111] Potential antagonists also include a small molecule which bindsto and occupies the active site of the polypeptides thereby making theminaccessible to substrate such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

[0112] The antagonists may be employed to treat leukemia, which resultsfrom oncogene activation in hemopoietic cells due to a chromosomaltranslocation. The polypeptides of the present invention may have adirect or indirect function in the activation of a cellular oncogeneresulting in leukemia.

[0113] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

Modes of Administration

[0114] The polypeptides and agonists or antagonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier. Such compositions comprise a therapeutically effective amountof the polypeptide, and a pharmaceutically acceptable carrier orexcipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The formulation should suit the mode of administration.

[0115] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides and agonists or antagonists of the presentinvention may be employed in conjunction with other therapeuticcompounds.

[0116] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intramuscular orsubcutaneous routes. The pharmaceutical compositions are administered inan amount which is effective for treating and/or prophylaxis of thespecific indication. In general, they are administered in an amount ofat least about 10 μg/kg body weight and in most cases they will beadministered in an amount not in excess of about 8 mg/Kg body weight perday. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kgbody weight daily, taking into account the routes of administration,symptoms, etc.

Gene Therapy

[0117] The polypeptides of the present invention, and agonists andantagonists which are polypeptides, may also be employed in accordancewith the present invention by expression of such polypeptides in vivo,which is often referred to as “gene therapy.”

[0118] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0119] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0120] Retroviruses from which the retroviral vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

[0121] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller et al., Biotechniques 7(9):980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

[0122] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0123] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1 (1990), pp. 5-14, which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0124] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Chromosome Assay

[0125] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0126] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0127] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0128] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases. For a review of this technique, see Vermaet al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988).

[0129] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0130] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0131] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Antibodies

[0132] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0133] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0134] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,Nature 256:495-497 (1975)), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4:72 (1983)), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.(1985), pp. 77-96).

[0135] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0136] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0137] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0138] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0139] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0140] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res. 8:4057 (1980).

[0141] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0142] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,Cold Spring Harbor, N.Y. (1989), p. 146). Unless otherwise provided,ligation may be accomplished using known buffers and conditions with 10units of T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolaramounts of the DNA fragments to be ligated.

[0143] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

[0144] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLE 1 Cloning and Expression of BTG-2 Using the BaculovirusExpression System

[0145] The DNA sequence encoding the full length BTG-2 protein, ATCC#97025, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ end sequences of the gene:

[0146] The 5′ primer has the sequence 5′CAGTGGATCCGCCACCATGCAGCTTGAAATCCAAGTAGCAC 3′ (SEQ ID No. 5) and containsa Bam HI restriction enzyme site (in bold) followed by 6 nucleotidesresembling an efficient signal for the initiation of translation ineukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) and justbehind the first 26 nucleotides of the BTG-2 gene (the initiation codonfor translation “ATG” is underlined).

[0147] The 3′ primer has the sequence 5′CAGTGGTACCATACATTTTCTTTTTTTTAGTTAGCCAT 3′ (SEQ ID No. 6) and containsthe cleavage site for the restriction endonuclease Asp718 and 25nucleotides complementary to the 3′ non-translated sequence of the BTG-2gene. The amplified sequences are isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment is then digested with the endonucleases Bam HI andAsp718 and purified again on a 1% agarose gel. This fragment isdesignated F2.

[0148] The vector pRG1 (modification of pVL941 vector, discussed below)is used for the expression of the BTG-2 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E., Amanual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555(1987)). This expression vector contains the strong polyhedrin promoterof the Autographa californica nuclear polyhidrosis virus (AcMNPV)followed by the recognition sites for the restriction endonucleases. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant viruses thebeta-galactosidase gene from E.coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofco-transfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology 170:31-39).

[0149] The plasmid is digested with the restriction enzymes Bam HI andAsp718 and dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA is then isolated from a 1% agarosegel using the commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated V2.

[0150] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. E. coli HB101 cells are then transformed and bacteriaidentified that contained the plasmid (pBac BTG-2) with the BTG-2 geneusing the respective restriction enzymes. The sequence of the clonedfragment is confirmed by DNA sequencing.

[0151] 5 μg of the plasmid pBac BTG-2 is co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al., Proc. Natl. Acad. Sci. (USA) 84:7413-7417 (1987)).

[0152] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac BTG-2are mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate is put back into an incubator and cultivation continued at 27°C. for four days.

[0153] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0154] Four days after the serial dilution the viruses are added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0155] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-BTG-2 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 2 Cloning and Expression of BTG-3 Using the BaculovirusExpression System

[0156] The DNA sequence encoding the full length BTG-3 protein, ATCC#97010, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ end sequences of the gene:

[0157] The 5′ primer has the sequence 5′CAGTGGATCCGCCACCATGCAGCTAGAGATCAAAGTGGCCC 3′ (SEQ ID No:7) and containsa Bam HI restriction enzyme site (in bold) followed by 6 nucleotidesresembling an efficient signal for the initiation of translation ineukaryotic cells (Kozak, M., J. Mol. Biol. 196:947-950 (1987)) and justbehind the first 228 nucleotides of the BTG-3 gene (the initiation codonfor translation “ATG” is underlined).

[0158] The 3′ primer has the sequence 5′CAGTGGTACCACGGGCCAGGTAGATGGTCAGTTGGCCAGCAC 3′ (SEQ ID No:8) and containsthe cleavage site for the restriction endonuclease Asp718 and 25nucleotides complementary to the 3′ non-translated sequence of the BTG-3gene. The amplified sequences are isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment is then digested with the endonucleases Bam HI andAsp718 and purified again on a 1% agarose gel. This fragment isdesignated F2.

[0159] The vector pRG1 (modification of pVL941 vector, discussed below)is used for the expression of the BTG-3 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases Bam HI andAsp718. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology170:31-39).

[0160] The plasmid is digested with the respective restriction enzymesand dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

[0161] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. E. coli HB101 cells are then transformed and bacteriaidentified that contained the plasmid (pBac BTG-3) with the BTG-3 geneusing the enzymes Bam HI and Asp718. The sequence of the cloned fragmentis confirmed by DNA sequencing.

[0162] 5 μg of the plasmid pBac BTG-3 is co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al., Proc. Natl. Acad. Sci. (USA) 84:7413-7417 (1987)).

[0163] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac BTG-3are mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace' medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with) 10% fetal calf serum is added.The plate is put back into an incubator and cultivation continued at 27°C. for four days.

[0164] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0165] Four days after the serial dilution the viruses are added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0166] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-BTG-3 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 3 Expression of Recombinant BTG-2 in COS Cells

[0167] The expression of plasmid, BTG-2 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire BTG-2 precursorand a HA tag fused in frame to its 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag correspond to an epitopederived from the influenza hemagglutinin protein as previously described(Wilson, I. et al., Cell 37:767 (1984)). The fusion of HA tag to thetarget protein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0168] The plasmid construction strategy is described as follows:

[0169] The DNA sequence encoding BTG-2, ATCC #97025, is constructed byPCR using two primers: the 5′ primer 5′GGCCCAAGCTTGCCGCCATGCAGCTTGAAATCCAAGTAG 3′ (SEQ ID No:9) contains a HindIII site followed by 23 nucleotides of BTG-2 coding sequence startingfrom the initiation codon; the 3′ sequence 5′ATCGTCTAGATTAGTTAGCCATAACAGGCTGGAATTGCTGGTTAGAATACTGCATGTTATTTAAGCTAAAATTCAAGCCATCTA 3′ (SEQ ID No:10) containscomplementary sequences to XbaI site, translation stop codon, HA tag andthe last 68 nucleotides of the BTG-2 coding sequence (not including thestop codon). Therefore, the PCR product contains a Hind III site, BTG-2coding sequence followed by HA tag fused in frame, a translationtermination stop codon next to the HA tag, and an XbaI site. The PCRamplified DNA fragment and the vector, pcDNAI/Amp, are digested withHind III and XbaI restriction enzyme and ligated. The ligation mixtureis transformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif.) the transformed culture is plated on ampicillin mediaplates and resistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant BTG-2, COS cellsare transfected with the expression vector by DEAE-DEXTRAN method(Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Theexpression of the BTG-2 HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1988)). Cells are labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media are then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5). Wilson, I. et al., Cell 37:767 (1984)). Bothcell lysate and culture media are precipitated with a HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

EXAMPLE 4 Expression of Recombinant BTG-3 in COS Cells

[0170] The expression of plasmid, BTG-3 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire BTG-3 precursorand a HA tag fused in frame to its 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag corresponds to an epitopederived from the influenza hemagglutinin protein as previously described(Wilson, I. et al., Cell 37:767 (1984)). The infusion of HA tag to thetarget protein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0171] The plasmid construction strategy is described as follows:

[0172] The DNA sequence encoding BTG-3, ATCC #97010, is constructed byPCR using two primers: the 5′ primer 5′GGCCCAAGCTTGCCGCCATGCAGCTAGAGATCAAAGTGGC 3′ (SEQ ID No. 11) contains aHind III site followed by 23 nucleotides of BTG-3 coding sequencestarting from the initiation codon; the 3′ sequence 5′ATCGTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGGTTGTAGCTGAGGCCTTCCACAAAGGGTGTCTTCTCCAGG 3′ (SEQ ID No:12) containscomplementary sequences to an XbaI site, translation stop codon, HA tagand the last 68 nucleotides of the BTG-3 coding sequence (not includingthe stop codon). Therefore, the PCR product contains a Hind III site,BTG-3 coding sequence followed by HA tag fused in frame, a translationtermination stop codon next to the HA tag, and an XbaI site. The PCRamplified DNA fragment and the vector, pcDNAI/Amp, are digested withHind III and XbaI restriction enzymes and ligated. The ligation mixtureis transformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif.) the transformed culture is plated on ampicillin mediaplates and resistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant BTG-3, COS cellsare transfected with the expression vector by DEAE-DEXTRAN method(Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Theexpression of the BTG-3 HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1988)). Cells are labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media is then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Cell 37:767 (1984)). Bothcell lysate and culture media are precipitated with a HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

EXAMPLE 5 Bacterial Expression and Purification of BTG-2

[0173] The DNA sequence encoding BTG-2, ATCC #97025, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end sequences of the processed BTG-2 gene (minus the signal peptidesequence) and the vector sequences 3′ to the BTG-2 gene. Additionalnucleotides corresponding to NcoI and Bgl II restriction enzyme siteswere added to the 5′ and 3′ end sequences respectively. The 5′oligonucleotide primer has the sequenceATCGCCATGGGACAGCTTGAAATCCAAGTAGCACTA 3′ (SEQ ID No:13) contains a Nco Irestriction enzyme site followed by 24 nucleotides of BTG-2 codingsequence. The 3′ sequence 5′ ATCGAGATCTTTAGTTAGCCATAACAGGCTGGAAT 3′ (SEQID No:14) contains complementary sequences to a Bgl II restriction siteand the last 21 nucleotides of BTG-2. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-60 (Qiagen, Inc. Chatsworth, Calif.). pQE-60 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. pQE-60 is thendigested with Nco I and Bgl II. The amplified sequences are ligated intopQE-60 and are inserted in frame with the sequence encoding for thehistidine tag and the RBS. The ligation mixture is then used totransform E. coli strain M15/rep 4 (Qiagen, Inc.) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989). M15/rep4 contains multiple copies of the plasmid pREP4, whichexpresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedBTG-2 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J Chromatography411:177-184 (1984)). BTG-2 (95% pure) is eluted from the column in 6molar guanidine HCl pH 5.0 and for the purpose of renaturation adjustedto 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein is dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 6 Bacterial Expression and Purification of BTG-3

[0174] The DNA sequence encoding BTG-3, ATCC #97010 is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end sequences of the processed BTG-3 gene. Additional nucleotidescorresponding to NcoI and Bgl II restriction enzyme sites are added tothe 5′ and 3′ sequences respectively. The 5′ oligonucleotide primer hasthe sequence 5′ ATCGCCATGGGACAGCTAGAGATCAAAGTGGCCCTG3′ (SEQ ID No:15)contains an NcoI restriction enzyme site followed by 24 nucleotides ofBTG-3 coding sequence. The 3′ sequence 5′ATCGAGATCTGTTGGCCAGCACCACGGGCTGG 3′ (SEQ ID NO.:16) containscomplementary sequences to a Bgl II restriction site and the last 21nucleotides of BTG-3 coding sequence. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-60 (Qiagen, Inc. Chatsworth, Calif.). pQE-60 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. pQE-60 is thendigested with Nco I and Bgl II. The amplified sequences are ligated intopQE-60 and are inserted in frame with the sequence encoding for thehistidine tag and the RBS. The ligation mixture is then used totransform E. coli strain M15/rep 4 (Qiagen, Inc.) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (160860). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 μg/ml) and Kan (25 μg/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedBTG-3 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J Chromatography411:177-184 (16084)). BTG-3 (95% pure) is eluted from the column in 6molar guanidine HCl pH 5.0 and for the purpose of renaturation adjustedto 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein is dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 7

[0175] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0176] pMV-7 (Kirschmeier, P. T. et al, DNA 7:219-25 (1988)) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0177] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified $EcoRIand HindIII fragment are added together, in the presence of T4 DNAligase. The resulting mixture is maintained under conditions appropriatefor ligation of the two fragments. The ligation mixture is used totransform bacteria HB101, which are then plated onto agar-containingkanamycin for the purpose of confirming that the vector had the gene ofinterest properly inserted.

[0178] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0179] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0180] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0181] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0182] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

[0183] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 18 1843 base pairs nucleic acid double linear DNA (genomic) CDS27..1061 1 GGCACGAGAT TTTGTGGCGT AGAGCT ATG CAG CTT GAA ATC CAA GTA GCACTA 53 Met Gln Leu Glu Ile Gln Val Ala Leu 1 5 AAT TTT ATT ATT TCG TATTTG TAC AAT AAG CTT CCC AGG AGA CGT GTC 101 Asn Phe Ile Ile Ser Tyr LeuTyr Asn Lys Leu Pro Arg Arg Arg Val 10 15 20 25 AAC ATT TTT GGT GAA GAACTT GAA AGA CTT CTT AAG AAG AAA TAT GAA 149 Asn Ile Phe Gly Glu Glu LeuGlu Arg Leu Leu Lys Lys Lys Tyr Glu 30 35 40 GGG CAC TGG TAT CCT GAA AAGCCA TAC AAA GGA TCG GGG TTT AGA TGT 197 Gly His Trp Tyr Pro Glu Lys ProTyr Lys Gly Ser Gly Phe Arg Cys 45 50 55 ATA CAC ATA GGG GAG AAA GTG GACCCA GTG ATT GAA CAA GCA TCC AAA 245 Ile His Ile Gly Glu Lys Val Asp ProVal Ile Glu Gln Ala Ser Lys 60 65 70 GAG AGT GGT TTG GAC ATT GAT GAT GTTCGT GGC AAT CTG CCA CAG GAT 293 Glu Ser Gly Leu Asp Ile Asp Asp Val ArgGly Asn Leu Pro Gln Asp 75 80 85 CTT AGT GTT TGG ATC GAC CCA TTT GAG GTTTCT TAC CAA ATT GGT GAA 341 Leu Ser Val Trp Ile Asp Pro Phe Glu Val SerTyr Gln Ile Gly Glu 90 95 100 105 AAG GGA CCA GTG AAG GTG CTT TAC GTGGAT GAT AAT AAT GAA AAT GGA 389 Lys Gly Pro Val Lys Val Leu Tyr Val AspAsp Asn Asn Glu Asn Gly 110 115 120 TGT GAG TTG GAT AAG GAG ATC AAA AACAGC TTT AAC CCA GAG GCC CAG 437 Cys Glu Leu Asp Lys Glu Ile Lys Asn SerPhe Asn Pro Glu Ala Gln 125 130 135 GTT TTT ATG CCC ATA AGT GAC CCA GCCTCA TCA GTG TCC AGC TCT CCA 485 Val Phe Met Pro Ile Ser Asp Pro Ala SerSer Val Ser Ser Ser Pro 140 145 150 TCG CCT CCT TTT GGT CAC TCT GCT GCTGTA AGC CCT ACC TTC ATG CCC 533 Ser Pro Pro Phe Gly His Ser Ala Ala ValSer Pro Thr Phe Met Pro 155 160 165 CGG TCC ACT CAG CCT TTA ACC TTT ACCACT GCC ACT TTT GCT GCC ACC 581 Arg Ser Thr Gln Pro Leu Thr Phe Thr ThrAla Thr Phe Ala Ala Thr 170 175 180 185 AAG TTC GGC TCT ACC AAA ATG AAAAAT AGT GGC CGT AGC AAC AAG GTT 629 Lys Phe Gly Ser Thr Lys Met Lys AsnSer Gly Arg Ser Asn Lys Val 190 195 200 GCA CGT ACT TCT CCC ATC AAC CTCGGC TTG AAT GTG AAT GAC CTC TTG 677 Ala Arg Thr Ser Pro Ile Asn Leu GlyLeu Asn Val Asn Asp Leu Leu 205 210 215 AAG CAG AAA GCC ATC TCT TCC TCAATG CAC TCT CTG TAT GGG CTT GGC 725 Lys Gln Lys Ala Ile Ser Ser Ser MetHis Ser Leu Tyr Gly Leu Gly 220 225 230 TTG GGT AGC CAG CAG CAG CCA CAGCAA CAG CAG CAG CCA GCC CAG CCG 773 Leu Gly Ser Gln Gln Gln Pro Gln GlnGln Gln Gln Pro Ala Gln Pro 235 240 245 CCA CCG CCA CCA CCA CCA CCA CAGCAG CAA CAA CAG CAG AAA ACC TCT 821 Pro Pro Pro Pro Pro Pro Pro Gln GlnGln Gln Gln Gln Lys Thr Ser 250 255 260 265 GCT CTT TCT CCT AAT GCC AAGGAA TTT ATT TTT CCT AAT ATG CAG GGT 869 Ala Leu Ser Pro Asn Ala Lys GluPhe Ile Phe Pro Asn Met Gln Gly 270 275 280 CAA GGT AGT AGT ACC AAT GGAATG TTC CCA GGT GAC AGC CCC CTT AAC 917 Gln Gly Ser Ser Thr Asn Gly MetPhe Pro Gly Asp Ser Pro Leu Asn 285 290 295 CTC AGT CCT CTC CAG TAC AGTAAT GCC TTT GAT GTG TTT GCA GCC TAT 965 Leu Ser Pro Leu Gln Tyr Ser AsnAla Phe Asp Val Phe Ala Ala Tyr 300 305 310 GGA GGC CTC AAT GAG AAG TCTTTT GTA GAT GGC TTG AAT TTT AGC TTA 1013 Gly Gly Leu Asn Glu Lys Ser PheVal Asp Gly Leu Asn Phe Ser Leu 315 320 325 AAT AAC ATG CAG TAT TCT AACCAG CAA TTC CAG CCT GTT ATG GCT AAC 1061 Asn Asn Met Gln Tyr Ser Asn GlnGln Phe Gln Pro Val Met Ala Asn 330 335 340 345 TAAAAAAAAG AAAATGTATCGTACAAGTTA AAATGCACGG GCCAAGGGGG GATTTTTT 1121 TTCACCTCCT TGAGAATTTTTTTTTTTAAG CTTATAGTAA GGATACATTC AAGCTTGG 1181 AAAAAAATAA TAATAAAACATGCATCATTT TTCATTTGCC AACCAAGCAC AAAGTTAT 1241 TATGCTGCCT GTATATTTTAAAGTATACTC TCAGATATGC CCTCTTACAG TATTTTAA 1301 TATTAGCAAA GGACATGGCTTGATTTTTTT TTATAAAAAT TGGCACTAAT AAGTGGGT 1361 ATTGGTCTTT TCTAATTGTATAATTTAATT TAGTACCAAA GTTTGTAAAA TATCAGAG 1421 TATATATATA TTGTATCCTACGACATGGTA TTGCATTTAT ATCTTTTTAC TACAGTGA 1481 TGTGACAGCA GCAGCCTCATGTTGTATTTT TTTTACTGAA ATTGTAAAAT ATCCATCT 1541 AAGACATCAA CTATTCTAAAAATTGTGTAC AGGATATTCC TTTAGTGGTG GAATTAAA 1601 GTGCGAATAC TTGCTTTCTCCAAAAAAATG TATTTTCTGT TAAAAGTTTA AAGATTTT 1661 CTATATATTA TGGAAGGAAAATGTAATCGT AAATATTAAT TTTGTACCTA TATTGTGC 1721 TACTTGAAAA AAACGGTATAAAAGTATTTT GAGTCAGTGT CTTACATGTT AAGAGGGA 1781 GAAATAGTTT ATATTAAGTTTGTATTAAAA TTCTTTAAAA TTAAAAAAAA AAAAAAAA 1841 AA 1843 345 amino acidsamino acid linear protein 2 Met Gln Leu Glu Ile Gln Val Ala Leu Asn PheIle Ile Ser Tyr Leu 1 5 10 15 Tyr Asn Lys Leu Pro Arg Arg Arg Val AsnIle Phe Gly Glu Glu Leu 20 25 30 Glu Arg Leu Leu Lys Lys Lys Tyr Glu GlyHis Trp Tyr Pro Glu Lys 35 40 45 Pro Tyr Lys Gly Ser Gly Phe Arg Cys IleHis Ile Gly Glu Lys Val 50 55 60 Asp Pro Val Ile Glu Gln Ala Ser Lys GluSer Gly Leu Asp Ile Asp 65 70 75 80 Asp Val Arg Gly Asn Leu Pro Gln AspLeu Ser Val Trp Ile Asp Pro 85 90 95 Phe Glu Val Ser Tyr Gln Ile Gly GluLys Gly Pro Val Lys Val Leu 100 105 110 Tyr Val Asp Asp Asn Asn Glu AsnGly Cys Glu Leu Asp Lys Glu Ile 115 120 125 Lys Asn Ser Phe Asn Pro GluAla Gln Val Phe Met Pro Ile Ser Asp 130 135 140 Pro Ala Ser Ser Val SerSer Ser Pro Ser Pro Pro Phe Gly His Ser 145 150 155 160 Ala Ala Val SerPro Thr Phe Met Pro Arg Ser Thr Gln Pro Leu Thr 165 170 175 Phe Thr ThrAla Thr Phe Ala Ala Thr Lys Phe Gly Ser Thr Lys Met 180 185 190 Lys AsnSer Gly Arg Ser Asn Lys Val Ala Arg Thr Ser Pro Ile Asn 195 200 205 LeuGly Leu Asn Val Asn Asp Leu Leu Lys Gln Lys Ala Ile Ser Ser 210 215 220Ser Met His Ser Leu Tyr Gly Leu Gly Leu Gly Ser Gln Gln Gln Pro 225 230235 240 Gln Gln Gln Gln Gln Pro Ala Gln Pro Pro Pro Pro Pro Pro Pro Pro245 250 255 Gln Gln Gln Gln Gln Gln Lys Thr Ser Ala Leu Ser Pro Asn AlaLys 260 265 270 Glu Phe Ile Phe Pro Asn Met Gln Gly Gln Gly Ser Ser ThrAsn Gly 275 280 285 Met Phe Pro Gly Asp Ser Pro Leu Asn Leu Ser Pro LeuGln Tyr Ser 290 295 300 Asn Ala Phe Asp Val Phe Ala Ala Tyr Gly Gly LeuAsn Glu Lys Ser 305 310 315 320 Phe Val Asp Gly Leu Asn Phe Ser Leu AsnAsn Met Gln Tyr Ser Asn 325 330 335 Gln Gln Phe Gln Pro Val Met Ala Asn340 345 1343 base pairs nucleic acid double linear DNA (genomic) CDS229..1260 3 GGAATTCGGC ACGAGCAACC CTCAACGACG AAAAGGACTT CGGTCCCCTGGCCCGGCGAC 60 GCCCGGGAAG GAAAGGAGAG CGACCTCCGC CCCGCGCTCA GGCCACCCTGGAGGGAGAA 120 CCGCCCCGCG CSSGSGTTAG AGCGCCCCGC CGCCCCGTAG ACCCGAAGCCGCCTGGAGC 180 CAAGGCTGTA CACGTGCCCT GTGCTGATTC TCTGCCTAGG AAAGGACC ATGCAG CTA 237 Met Gln Leu 1 GAG ATC AAA GTG GCC CTG AAC TTC ATC ATC TCCTAC TTG TAC AAC AAG 285 Glu Ile Lys Val Ala Leu Asn Phe Ile Ile Ser TyrLeu Tyr Asn Lys 5 10 15 CTG CCC CGG CGC CGG GCA GAC CTG TTT GGG GAG GAGCTA GAG CGG CTT 333 Leu Pro Arg Arg Arg Ala Asp Leu Phe Gly Glu Glu LeuGlu Arg Leu 20 25 30 35 TTG AAA AGG AAA TAT GAA GGC CAC TGG TAC CCT GAGAAG CCA CTG AAA 381 Leu Lys Arg Lys Tyr Glu Gly His Trp Tyr Pro Glu LysPro Leu Lys 40 45 50 GGC TCT GGC TTC CGC TGT GTT CAC ATT GGG GAG ATG GTGGAC CCC GTG 429 Gly Ser Gly Phe Arg Cys Val His Ile Gly Glu Met Val AspPro Val 55 60 65 GTG GAG CTG GCC GCC AAG CGG AGT GGC CTG GCG GTG GAA GATGTG CGG 477 Val Glu Leu Ala Ala Lys Arg Ser Gly Leu Ala Val Glu Asp ValArg 70 75 80 GCC AAT GTG CCT GAG GAG CTG AGT GTC TGG ATT GAT CCC TTT GAGGTG 525 Ala Asn Val Pro Glu Glu Leu Ser Val Trp Ile Asp Pro Phe Glu Val85 90 95 TCC TAC CAG ATT GGT GAG AAG GGA GCT GTG AAA GTG CTG TAC CTG GAT573 Ser Tyr Gln Ile Gly Glu Lys Gly Ala Val Lys Val Leu Tyr Leu Asp 100105 110 115 GAC AGT GAG GGT TGC GGT GCC CCA GAG CTG GAC AAG GAG ATC AAGAGC 621 Asp Ser Glu Gly Cys Gly Ala Pro Glu Leu Asp Lys Glu Ile Lys Ser120 125 130 AGC TTC AAC CCT GAC GCC CAG GTG TTC GTG CCC ATT GGC AGC CAGGAC 669 Ser Phe Asn Pro Asp Ala Gln Val Phe Val Pro Ile Gly Ser Gln Asp135 140 145 AGC TCC CTG TCC AAC TCC CCA TCG CCA TCC TTT GGC CAG TCA CCCAGC 717 Ser Ser Leu Ser Asn Ser Pro Ser Pro Ser Phe Gly Gln Ser Pro Ser150 155 160 CCT ACC TTC ATT CCC CGC TCC GCT CAG CCC ATC ACC TTC ACC ACCGCC 765 Pro Thr Phe Ile Pro Arg Ser Ala Gln Pro Ile Thr Phe Thr Thr Ala165 170 175 TCC TTC GCT GCC ACC AAA TTT GGC TCC ACT AAG ATG AAG AAG GGGGGC 813 Ser Phe Ala Ala Thr Lys Phe Gly Ser Thr Lys Met Lys Lys Gly Gly180 185 190 195 GGG GCA GCA AGT GGT GGG GGT GTA GCC AGC AGT GGG GCG GGTGGC CAG 861 Gly Ala Ala Ser Gly Gly Gly Val Ala Ser Ser Gly Ala Gly GlyGln 200 205 210 CAG CCA CCA CAG CAG CCT CGC ATG GCC CGC TCA CCC ACC AACAGC CTG 909 Gln Pro Pro Gln Gln Pro Arg Met Ala Arg Ser Pro Thr Asn SerLeu 215 220 225 CTG AAG CAC AAG AGC CTC TCT CTG TCT ATG CAT TCA CTG AACTTC ATC 957 Leu Lys His Lys Ser Leu Ser Leu Ser Met His Ser Leu Asn PheIle 230 235 240 ACG GCC AAC CCG GCC CCT CAG TCC CAG CTC TCA CCC AAT GCCAAG GAG 1005 Thr Ala Asn Pro Ala Pro Gln Ser Gln Leu Ser Pro Asn Ala LysGlu 245 250 255 TTC GTG TAC AAC GGT GGT GGC TCA CCC AGC CTC TTC TTT GATGCG GCC 1053 Phe Val Tyr Asn Gly Gly Gly Ser Pro Ser Leu Phe Phe Asp AlaAla 260 265 270 275 GAT GGC CAG GGC AGC GGC ACC CCA GGC CCG TTT GGA GGCAGT GGG GCT 1101 Asp Gly Gln Gly Ser Gly Thr Pro Gly Pro Phe Gly Gly SerGly Ala 280 285 290 GGC ACC TGC AAC AGC AGC AGC TTT GAC ATG GCC CAG GTATTT GGA GGT 1149 Gly Thr Cys Asn Ser Ser Ser Phe Asp Met Ala Gln Val PheGly Gly 295 300 305 GGT GCC AAC AGC CTC TTC CTG GAG AAG ACA CCC TTT GTGGAA GGC CTC 1197 Gly Ala Asn Ser Leu Phe Leu Glu Lys Thr Pro Phe Val GluGly Leu 310 315 320 AGC TAC AAC CTG AAC ACC ATG CAG TAT CCC AGC CAG CAGTTC CAG CCC 1245 Ser Tyr Asn Leu Asn Thr Met Gln Tyr Pro Ser Gln Gln PheGln Pro 325 330 335 GTG GTG CTG GCC AAC TGACCATCTA CCTGGCCCGT GGGGGCAGGAGCACCCAAGA 1300 Val Val Leu Ala Asn 340 CCACAGAAAA GAGAAAGGAA AGGCCAAAAAAAAAAAAAAA AAA 1343 344 amino acids amino acid linear protein 4 Met GlnLeu Glu Ile Lys Val Ala Leu Asn Phe Ile Ile Ser Tyr Leu 1 5 10 15 TyrAsn Lys Leu Pro Arg Arg Arg Ala Asp Leu Phe Gly Glu Glu Leu 20 25 30 GluArg Leu Leu Lys Arg Lys Tyr Glu Gly His Trp Tyr Pro Glu Lys 35 40 45 ProLeu Lys Gly Ser Gly Phe Arg Cys Val His Ile Gly Glu Met Val 50 55 60 AspPro Val Val Glu Leu Ala Ala Lys Arg Ser Gly Leu Ala Val Glu 65 70 75 80Asp Val Arg Ala Asn Val Pro Glu Glu Leu Ser Val Trp Ile Asp Pro 85 90 95Phe Glu Val Ser Tyr Gln Ile Gly Glu Lys Gly Ala Val Lys Val Leu 100 105110 Tyr Leu Asp Asp Ser Glu Gly Cys Gly Ala Pro Glu Leu Asp Lys Glu 115120 125 Ile Lys Ser Ser Phe Asn Pro Asp Ala Gln Val Phe Val Pro Ile Gly130 135 140 Ser Gln Asp Ser Ser Leu Ser Asn Ser Pro Ser Pro Ser Phe GlyGln 145 150 155 160 Ser Pro Ser Pro Thr Phe Ile Pro Arg Ser Ala Gln ProIle Thr Phe 165 170 175 Thr Thr Ala Ser Phe Ala Ala Thr Lys Phe Gly SerThr Lys Met Lys 180 185 190 Lys Gly Gly Gly Ala Ala Ser Gly Gly Gly ValAla Ser Ser Gly Ala 195 200 205 Gly Gly Gln Gln Pro Pro Gln Gln Pro ArgMet Ala Arg Ser Pro Thr 210 215 220 Asn Ser Leu Leu Lys His Lys Ser LeuSer Leu Ser Met His Ser Leu 225 230 235 240 Asn Phe Ile Thr Ala Asn ProAla Pro Gln Ser Gln Leu Ser Pro Asn 245 250 255 Ala Lys Glu Phe Val TyrAsn Gly Gly Gly Ser Pro Ser Leu Phe Phe 260 265 270 Asp Ala Ala Asp GlyGln Gly Ser Gly Thr Pro Gly Pro Phe Gly Gly 275 280 285 Ser Gly Ala GlyThr Cys Asn Ser Ser Ser Phe Asp Met Ala Gln Val 290 295 300 Phe Gly GlyGly Ala Asn Ser Leu Phe Leu Glu Lys Thr Pro Phe Val 305 310 315 320 GluGly Leu Ser Tyr Asn Leu Asn Thr Met Gln Tyr Pro Ser Gln Gln 325 330 335Phe Gln Pro Val Val Leu Ala Asn 340 41 base pairs nucleic acid singlelinear cDNA 5 CAGTGGATCC GCCACCATGC AGCTTGAAAT CCAAGTAGCA C 41 38 basepairs nucleic acid single linear cDNA 6 CAGTGGTACC ATACATTTTC TTTTTTTTAGTTAGCCAT 38 41 base pairs nucleic acid single linear cDNA 7 CAGTGGATCCGCCACCATGC AGCTAGAGAT CAAAGTGGCC C 41 42 base pairs nucleic acid singlelinear cDNA 8 CAGTGGTACC ACGGGCCAGG TAGATGGTCA GTTGGCCAGC AC 42 39 basepairs nucleic acid single linear cDNA 9 GGCCCAAGCT TGCCGCCATG CAGCTTGAAATCCAAGTAG 39 84 base pairs nucleic acid single linear cDNA 10 ATCGTCTAGATTAGTTAGCC ATAACAGGCT GGAATTGCTG GTTAGAATAC TGCATGTTAT 60 TTAAGCTAAAATTCAAGCCA TCTA 84 40 base pairs nucleic acid single linear cDNA 11GGCCCAAGCT TGCCGCCATG CAGCTAGAGA TCAAAGTGGC 40 81 base pairs nucleicacid single linear cDNA 12 ATCGTCTAGA TCAAGCGTAG TCTGGGACGT CGTATGGGTAGGTTGTAGCT GAGGCCTTCC 60 ACAAAGGGTG TCTTCTCCAG G 81 36 base pairsnucleic acid single linear cDNA 13 ATCGCCATGG GACAGCTTGA AATCCAAGTAGCACTA 36 35 base pairs nucleic acid single linear cDNA 14 ATCGAGATCTTTAGTTAGCC ATAACAGGCT GGAAT 35 36 base pairs nucleic acid single linearcDNA 15 ATCGCCATGG GACAGCTAGA GATCAAAGTG GCCCTG 36 32 base pairs nucleicacid single linear cDNA 16 ATCGAGATCT GTTGGCCAGC ACCACGGGCT GG 32 171amino acids amino acid not relevant linear protein 17 Met His Pro PheTyr Thr Arg Ala Ala Thr Met Ile Gly Glu Ile Al 1 5 10 15 Ala Ala Val SerPhe Ile Ser Lys Phe Leu Arg Thr Lys Gly Leu Th 20 25 30 Ser Glu Arg GlnLeu Gln Thr Phe Ser Gln Ser Leu Gln Glu Leu Le 35 40 45 Ala Glu His TyrLys His His Trp Phe Pro Glu Lys Pro Cys Lys Gl 50 55 60 Ser Gly Tyr ArgCys Ile Arg Ile Asn His Lys Met Asp Pro Leu Il 65 70 75 80 Gly Gln AlaAla Gln Arg Ile Gly Leu Ser Ser Gln Glu Leu Phe Ar 85 90 95 Leu Leu ProSer Glu Leu Thr Leu Trp Val Asp Pro Tyr Glu Val Se 100 105 110 Tyr ArgIle Gly Glu Asp Gly Ser Ile Cys Val Leu Tyr Glu Ala Se 115 120 125 ProAla Gly Gly Ser Thr Gln Asn Ser Thr Asn Val Gln Met Val As 130 135 140Ser Arg Ile Ser Cys Lys Glu Glu Leu Leu Leu Gly Arg Thr Ser Pr 145 150155 160 Ser Lys Asn Tyr Asn Met Met Thr Val Ser Gly 165 170 213 aminoacids amino acid not relevant linear peptide 18 Met Gln Leu Glu Ile ValAla Leu Asn Phe Ile Ile Ser Tyr Leu Ty 1 5 10 15 Asn Lys Leu Pro Arg ArgArg Phe Gly Glu Glu Leu Glu Arg Leu Le 20 25 30 Lys Lys Tyr Glu Gly HisTrp Tyr Pro Glu Lys Pro Lys Gly Ser Gl 35 40 45 Phe Arg Cys Ile His IleGly Glu Lys Val Asp Pro Val Ile Glu Gl 50 55 60 Ala Ala Lys Arg Ser GlyLeu Asp Val Arg Asn Leu Pro Glu Leu Se 65 70 75 80 Val Trp Ile Asp ProPhe Glu Val Ser Tyr Gln Ile Gly Glu Lys Gl 85 90 95 Val Lys Val Leu TyrAsp Asp Gly Gly Glu Leu Asp Lys Glu Ile Ly 100 105 110 Ser Ser Phe AsnPro Ala Gln Val Phe Pro Ile Ser Ser Ser Ser Pr 115 120 125 Ser Pro PheGly Ser Ser Pro Thr Phe Pro Arg Ser Gln Pro Thr Ph 130 135 140 Thr ThrAla Phe Ala Ala Thr Lys Phe Gly Ser Thr Lys Met Lys Gl 145 150 155 160Gly Asn Leu Leu Lys Lys Ser Ser Met His Ser Leu Gln Gln Leu Se 165 170175 Pro Asn Ala Lys Glu Phe Phe Gly Gln Gly Ser Thr Gly Phe Gly As 180185 190 Ser Phe Ala Leu Phe Val Gly Leu Leu Asn Met Gln Tyr Gln Gln Ph195 200 205 Gln Pro Val Ala Asn 210

What at is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding the BTG-2 or BTG-3 polypeptide having the completeamino acid sequence in FIG. 1 (SEQ ID NO:2) and FIG. 2 (SEQ ID NO:4),respectively; (b) a nucleotide sequence encoding the mature BTG-2 ormature BTG-3 polypeptide having the amino acid sequence at positionsfrom about 26 to about 345 in FIG. 1 (SEQ ID NO:2) and from about 19 toabout 344 in FIG. 2 (SEQ ID NO:4), respectively; (c) a nucleotidesequence encoding the BTG-2 or BTG-3 polypeptide having the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97025 and 97010, respectively; (d) a nucleotide sequence encodingthe mature BTG-2 or mature BTG-3 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97025and 97010, respectively; and (e) a nucleotide sequence complementary toany of the nucleotide sequences in (a), (b), (c) or (d).
 2. The nucleicacid molecule of claim 1 wherein said BTG-2 or BTG-3 polynucleotide hasthe complete nucleotide sequence in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQID NO:3), respectively.
 3. The nucleic acid molecule of claim 1 whereinsaid polynucleotide has the nucleotide sequence in FIG. 1 (SEQ ID NO:1)and FIG. 2 (SEQ ID NO:3) encoding the BTG-2 or BTG-3 polypeptide havingthe complete amino acid sequence in FIG. 1 (SEQ ID NO:2) and FIG. 2 (SEQID NO:4), respectively.
 4. The nucleic acid molecule of claim 1 whereinsaid polynucleotide has the nucleotide sequence in FIG. 1 (SEQ ID NO:1)and FIG. 2 (SEQ ID NO:3) encoding the mature BTG-2 or BTG-3 polypeptidehaving the amino acid sequence in FIG. 1 (SEQ ID NO:2) and FIG. 2 (SEQID NO:4), respectively.
 5. The nucleic acid molecule of claim 1 whereinsaid polynucleotide has the complete nucleotide sequence of the cDNAclone contained in ATCC Deposit No. 97025 or
 97010. 6. The nucleic acidmolecule of claim 1 wherein said polynucleotide has the nucleotidesequence encoding the BTG-2 or BTG-3 polypeptide having the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97025 and 97010, respectively.
 7. The nucleic acid molecule of claim1 wherein said polynucleotide has the nucleotide sequence encoding themature BTG-2 or BTG-3 polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97025 and 97010,respectively.
 8. An isolated nucleic acid molecule comprising apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence identical to anucleotide sequence in (a), (b), (c), (d) or (e) of claim 1 wherein saidpolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues.
 9. Anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a BTG-2 orBTG-3 polypeptide having an amino acid sequence in (a), (b), (c) or (d)of claim
 1. 10. A method for making a recombinant vector comprisinginserting an isolated nucleic acid molecule of claim 1 into a vector.11. A recombinant vector produced by the method of claim
 10. 12. Amethod of making a recombinant host cell comprising introducing therecombinant vector of claim 11 into a host cell.
 13. A recombinant hostcell produced by the method of claim
 12. 14. A recombinant method forproducing a BTG-2 or BTG-3 polypeptide, comprising culturing therecombinant host cell of claim 13 under conditions such that saidpolypeptide is expressed and recovering said polypeptide.
 15. Anisolated BTG-2 or BTG-3 polypeptide having an amino acid sequence atleast 95% identical to a sequence selected from the group consisting of:(a) the amino acid sequence of the BTG-2 or BTG-3 polypeptide having thecomplete 345 and 344 amino acid sequences shown in FIG. 1 (SEQ ID NO:2)and FIG. 2 (SEQ ID NO:4), respectively; (b) the amino acid sequence ofthe predicted mature BTG-2 or BTG-3 polypeptide (without the leader)having the amino acid sequence at positions from about 26 to about 345in FIG. 1 (SEQ ID NO:2) and from about 19 to about 344 in FIG. 2 (SEQ IDNO:4), respectively; (c) the amino acid sequence of the BTG-2 or BTG-3polypeptide having the complete amino acid sequence, including theleader, encoded by the cDNA clone contained in ATCC Deposit No. 97025and 97010, respectively; (d) the amino acid sequence of the mature BTG-2or BTG-3 polypeptide having the amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97025 and 97010, respectively; and(e) the amino acid sequence of an epitope-bearing portion of any one ofthe polypeptides of (a), (b), (c), or (d).
 16. An isolated antibody thatbinds specifically to a BTG-2 or BTG-3 polypeptide of claim
 15. 17. Amethod for the treatment of a patient having need of a polypeptide ofclaim 15 comprising administering a therapeutically effective amount ofthe polypeptide as claimed in claim
 15. 18. A method for the treatmentof a patient having need to inhibit a polypeptide of claim 15 comprisingadministering a therapeutically effective amount of the polypeptide asclaimed in claim
 15. 19. A method of claim 17 wherein saidtherapeutically effective amount of the polypeptide of claim 15 isadministered by providing the patient DNA encoding said polypeptide andexpressing said polypeptide in vivo.
 20. A process for diagnosing in apatient a disease or a susceptibility to a disease related to an alteredlevel of expression of the polypeptide of claim 15 comprisingdetermining a mutation in the nucleic acid sequence encoding saidpolypeptide in a sample derived from a patient.